248
Oxygen radicals and signaling
Toren Finkel
Recent evidence suggests that reactive oxygen species, such
as superoxide anions and hydrogen peroxide, function as
intracellular second messengers. This review will discuss the
progress in understanding the intracellular pathways leading
from ligand stimulation to the generation of oxidants, as well
as some of the increasing number of cellular processes that
appear to be subject to redox regulation.
Address
Cardiology Branch, National Institutes of Health, Building 10, Room
7B15, 10 Center Drive, Bethesda, MD 20892-1650, USA
Current Opinion in Cell Biology 1998,10:248–253
http://biomednet.com.elecref/0955067401000248
 Current Biology Ltd ISSN 0955-0674
Abbreviations
ERK
extracellular-signal-regulated kinase
JNK
c-Jun amino-terminal kinase
MAPK
mitogen-activated protein kinase
PDGF
platelet-derived growth factor
PKC
protein kinase C
ROS
reactive oxygen species
Introduction
Few biological entities have as bad a reputation as reactive
oxygen species. For many years, these small diffusible
molecules have been thought of as the unwanted and
toxic by-products of living in an aerobic environment.
Although the cell had clearly evolved multiple defenses for
their elimination, their relentless production coupled with
their damaging nature has led to the widely held belief
that these molecules serve only a harmful function. The
purpose of this review is to re-evaluate this prejudice and
to provide a summary of some recent evidence suggesting
that the production of reactive oxygen species (ROS) is
tightly regulated and serves a physiological function.
The notion that molecules such as superoxide anion
(O2−) and hydrogen peroxide could function in signal
transduction in mammalian cells is not without precedent.
Indeed, a wealth of information suggests that these
molecules function in this fashion in both bacteria and
plants. In bacteria, redox regulation of transcription occurs,
with a different set of genes stimulated by H2O2 and
O2− [1,2]. Similarly in the plant pathogen response, there
appears to be a clear role for H2O2 as a signaling
molecule [3].
In mammalian cells, the physiological role for O2− and
H2O2 is less well characterized than that of another
reactive oxygen species, namely nitric oxide. Analysis of
the role of NO suggests it functions in two discrete
fashions. Production of nitric oxide by macrophages and
other immune-effector cells results in the high level
production of NO, consistent with its role in host
defense. In contrast, the nitric oxide synthase found
in endothelial cells or neurons generates two to three
orders of magnitude less NO when activated. Produced
at this level, NO is widely believed to function in signal
transduction. This dichotomy between immune function
and signal transduction is likely to be preserved for other
reactive oxygen species. As seen in Figure 1, O2− and
H2O2 are produced in large amounts by cells of the
immune system. In contrast, other cell types, including
vascular smooth muscle cells, chondrocytes and fibroblasts
appear to produce significantly lower amounts of these
molecules. Emerging evidence suggests that this mini
‘oxidative burst’ appears to have an important role in signal
transduction.
Figure 1
[high]
NO
[low]
Immune
Signal
effector
transduction
Macrophage
Neurons/
endothelium
[high]
O2–/H2O2
[low]
Immune
? Signal
effector
transduction
Neutrophils
SMC/
chondrocytes/
fibroblasts
Current Opinion in Cell Biology
A potential analogy between nitric oxide and other reactive oxygen
species. In both cases, high levels are produced by immune effector
cells while lower amounts are used by other cell types for signal
transduction.
ROS as second messengers in signal
transduction
Studies over the past 10 years have demonstrated that
ligand stimulation of non-phagocytic cells results in an
increase in intracellular reactive oxygen species. This
phenomenon has been observed in a wide variety of
cell types and is stimulated by a diverse collection of
ligands, including cytokines [4–6] as well as peptide
growth factors acting through tyrosine kinase [6–8,9•] and
G-protein-coupled receptors [10].
The importance of this rise in ROS following ligand
activation has been appreciated only more recently.
Oxygen radicals and signaling Finkel
Analysis in vascular smooth muscle cells demonstrated
that stimulation by platelet-derived growth factor (PDGF)
results in a rapid increase in ROS which peaks within
minutes of ligand stimulation and then returns to baseline
[8]. This time course is similar to the time course of
growth-factor-stimulated tyrosine phosphorylation. The
link between ligand-stimulated H2O2 production and
phosphorylation was further strengthened by the observation that exogenous H2O2 mimics growth-factor-induced
tyrosine phosphorylation. In addition, increasing the level
of the peroxide-scavenging enzyme catalase blunted the
increase in H2O2 and inhibited the ability of PDGF to
stimulate tyrosine phosphorylation. Similar results were
obtained in A431 cells stimulated with epidermal growth
factor (EGF) [9•], again suggesting that ligand-stimulated
ROS generation may have a general role in mediating
tyrosine phosphorylation.
Although these results suggest that ROS may function
as a second messenger system in the context of ligand
stimulation, other evidence suggest that oxidative stress
may also activate unique pathways. The transcription
factor NF-κB is stimulated by a host of ligands leading
eventually to the serine phosphorylation and subsequent
proteosomal degradation of the IκB inhibitory subunit.
ROS are important in ligand-stimulated NF-κB activation
since most, if not all, such activation can be blocked by
antioxidant treatment [11]. Nonetheless, direct oxidative
stress such as hypoxic reoxygenation also activates NF-κB,
but appears to do so through a unique proteolysis
independent pathway involving tyrosine phosphorylation
[12•]. Similarly, addition of H2O2 appears to activate
various protein kinase C (PKC) family members [13•].
However, the activation of PKC by oxidants appears to be
independent of lipid cofactors and thereby differs from the
classical ligand-stimulated pathway.
not direct targets of ROS. Although not proven, one
such direct target may be tyrosine phosphatase. All such
molecules have in the active site a cysteine residue
which is essential for biological activity [20] and which
can be regulated in a redox-dependent manner [21].
This observation may provide a mechanistic explanation
linking H2O2 generation and tyrosine phosphorylation.
As demonstrated in Figure 2a, under basal conditions
when ROS levels are low, tyrosine phosphatase activity
would predominate since the specific activity of tyrosine
phosphatase is several orders of magnitude greater than
the corresponding activity of tyrosine kinases. Ligand
stimulation would result in an increase in ROS which
would function to transiently inactivate the activity of
tyrosine phosphatase (Figure 2b). When ROS levels
fell, phosphatase activity could be restored presumably
by cellular reducing enzymes. Under such a scenario,
growth-factor-stimulated ROS production would temporarily permit a burst of kinase activity through the transient
inactivation of phosphatases. Recent evidence suggests
that such a mechanism may also be common to other
non-classical activators of receptor tyrosine kinase activity
such as radiation and alkylating agents [22•].
Figure 2
(a)
(b)
Oxidase
Oxidase
Tyrosine
phosphatase
activity
O2
Although the activity of the extracellular-signal-regulated
kinase (ERKs) are redox sensitive, they are probably
O2–
H2O2
Tyrosine
kinase
activity
Targets of ROS
The downstream targets of ROS have remained largely
unexplored. Extracellular administration of non-lethal
concentrations of H2O2 has been demonstrated to activate
mitogen-activated protein kinase (MAPK) as well as the
c-Jun amino-terminal kinase (JNK) [8,14,15]. In addition
to the effects of exogenous ROS, the ability of ligands
to activate MAPK has been shown in certain examples to
be inhibited by treating cells with chemical or enzymatic
antioxidants [8,16]. This has also been demonstrated
for JNK activation, where it has been noted in several
cell types that activation of the kinase is inhibited
by pretreatment with the antioxidant N-acetylcysteine
[17•,18]. Since this antioxidant affects the level of
intracellular glutathione, it is consistent with other results
suggesting that some, but not all, ligands that activate JNK
are exquisitively sensitive to the intracellular glutathione
status of the cell [19].
249
Tyrosine
kinase
activity
Tyrosine
phosphatase
activity
Current Opinion in Cell Biology
A model for how ROS may regulate tyrosine phosphorylation.
(a) Under basal conditions, ROS levels are low and the specific
activity of tyrosine phosphatase exceeds that of the corresponding
kinases. (b) Following ligand stimulation, ROS levels increase,
perhaps as in the case of the neutrophil, through the recruitment
of cytosolic proteins to a membrane bound oxidase. Increase ROS
levels react with the redox-sensitive cysteine residues of tyrosine
phosphatases leading to their transient inactivation. This leads
to a burst of unopposed kinase activity until ROS levels fall and
phosphatase activity can be restored through reduction of the
oxidized cysteine residue.
The role of small GTP-binding proteins
The intracellular pathway in non-phagocytic cells leading
from ligand activation to ROS generation appears to share
some similarity to the better characterized neutrophil
system. In phagocytic cells, the small GTP-binding
protein Rac2 appears to have an important role in oxidase
function [23]. Similarly, a requirement for small GTPases,
250
Cell regulation
including Ras and Rac1, has been recently shown for ROS
generation following stimulation by cytokines and growth
factors [24•]. This study also demonstrated that expression
of constitutively active mutants of ras or rac1 lead to
an increase in levels of ROS. Genetic evidence suggests
that Rac1 acts downstream of Ras since expression of
a dominant-negative rac1 mutant inhibits Ras-stimulated
ROS production [24•].
Surprisingly, although the small GTP-binding Ras and
Rac1 may contribute to ROS production, they may themselves also be an important target of redox modulation.
Recent experiments have demonstrated that a cysteine
residue at position 118 of Ras can be regulated in a
redox-dependent fashion [25•]. Redox modification of
the residue appears to effect nucleotide exchange, and
conversion of cysteine 118 to serine results in an inhibition
of certain aspects of Ras-dependent signaling [25•].
The source of ROS
The enzymatic source or sources of ligand-stimulated ROS
have remained elusive. A variety of cellular enzymes, including cyclooxygenases and lipoxygenases, are potential
ligand-activated superoxide-generating systems. Perhaps
the most intriguing possibility is that by analogy with the
neutrophil system, an NADPH oxidase will be the primary
source of ROS involved in signaling. Such a hypothesis
is supported by the observation that a ligand-activated
enzyme with NADPH/NADH oxidase activity appears to
be present in a variety of non-phagocytic cells including
smooth muscle cells [10], chondrocytes [6], and kidney
epithelium [18]. Treatment with diphenyleneiodonium, a
pharmacological inhibitor of the flavoprotein component of
the neutrophil NADPH oxidase, appears to significantly
effect ROS production in non-phagocytic cells stimulated
by either ligands [6,10] or expression of activated small
GTPases [24•]. In addition, components of the NADPH
oxidase appear to be present in other cell types [26].
Inhibiting the expression of one of these ubiquitously
expressed components, p22phox, was recently shown
to inhibit the ability of angiotensin II to stimulate
superoxide production in vascular smooth muscle cells
[27•]. Cells deficient in p22phox no longer hypertrophied in
response to angiotensin II, again suggesting a physiological
downstream role for ROS.
ROS in growth and death
The observation that the small GTP-binding proteins Ras
and Rac regulate ROS production in non-phagocytic cells
may be important in understanding the role of these
proteins in growth control and transformation. Previous
experiments have demonstrated that treatment of some
cells with oxidant stress stimulated cell division and
the expression of growth-related gene products [28,29].
Recent experiments with Ras-transformed NIH 3T3
cells demonstrated an increase in superoxide production
compared to non-transformed or Raf-transformed 3T3
cells [30••]. The increase in O2− was inhibited by
dominant-negative Rac1 expression. Treatment with an
antioxidant inhibited S-phase progression in serum-starved
Ras-transformed cells but not in Raf-transformed cells.
These effects were independent of MAPK or JNK activation suggesting that oxidants may mediate a novel Rasdependent pathway important for cell-cycle progression
and transformation. Recent evidence further suggests that
this redox-dependent, small GTPase-regulated pathway
may have distinct and as yet uncharacterized downstream
targets. In particular, a mutant of Rac, defective for
superoxide production but still capable of activating
both JNK and cytoskeletal reorganization appears to be
unable to stimulate cell proliferation (D Bar-Sagi, personal
communication).
Although ROS may mediate growth regulatory pathways,
there is emerging, although sometimes conflicting, evidence that also suggests a role for ROS in apoptotic
pathways [31]. As apoptosis is triggered by multiple
agents and proceeds through multiple pathways it is
likely that ROS may participate in some, but not all,
aspects of programmed cell death. In this regard, it
has been recently observed that stimulation with Fas
ligand resulted in apoptosis through the generation of
O2− [32]. This process was inhibited by expression of a
dominant-negative ras gene, again suggesting a role for
small GTPases in the control of the redox state of the
cell. In addition, two recent studies have suggested that
ROS may mediate p53-dependent apoptosis [33•,34••].
Both studies employed adenovirus-mediated gene transfer
of wild-type p53. Overexpression of p53 resulted in a
significant increase in ROS levels, while treatment of cells
with antioxidants inhibited p53-mediated apoptosis. In
addition, analysis of gene products induced via p53-dependent transcriptional activation included a number of
proteins that function to regulate the intracellular redox
state [34••].
Redox regulation of transcription
Reactive oxygen species may directly regulate the activity
of transcription factors. As discussed previously, perhaps
the most widely studied example is the activation of
NF-κB. Activation of this factor, which can result from
stimulation by diverse agents, appears to proceed through
a common pathway involving ROS generation [11,35].
Using cells that overexpress either superoxide dismutase
or catalase, H2O2 and not O2− has been demonstrated to
be the relevant ROS [36]. Such specificity is reminiscent of
the SoxRS and OxyR system of bacteria. Consistent with
previous studies, the ligand-stimulated pathway leading
to H2O2 production and subsequent redox activation of
NF-κB has recently been shown to involve the Rho family
of small GTP-binding proteins [37,38].
Another transcription factor whose activity appears to be
regulated in a redox-dependent fashion is the hypoxia
inducible factor, Hif-1 [39]. Studies over the few several
years have demonstrated that the level of Hif-1 protein
Oxygen radicals and signaling Finkel
increases under hypoxic conditions. Interestingly, treatment of cells with H2O2 before hypoxia inhibits the
subsequent increase in Hif-1 levels [40,41]. Recent studies
have shown that one reason for the difference in Hif-1
levels between hypoxic and normoxic conditions is that,
in normoxic conditions, Hif-1 is subjected to degradation
through the ubiquitin pathway [42]. One explanation may
be that ambient levels of H2O2, which are higher under
normoxic conditions than during hypoxia, may regulate
ubiquitin activity. Such speculation is supported by some
recent observations that ubiquitin conjugation activity
could increase almost 10-fold in activity following a H2O2
challenge [43]. Alternatively, as has been suggested, ROS
may alter Hif-1, perhaps through phosphorylation, and
thereby alter its degradation rate [42].
Finally, the binding of certain transcription factors to
DNA appears to be regulated in a redox-dependent
fashion through the oxidation–reduction of critical cysteine
residues in the DNA-binding domain [44]. This area
was considerably aided by the isolation of Ref-1 [45],
a nuclear protein which appears to facilitate DNA
binding by specifically reducing cysteine residues in the
DNA-binding domain. Ref-1 activity is in turn regulated
by the antioxidant protein thioredoxin [42,46]. Agents that
induce oxidative stress, such as phorbol ester, appear to
cause the translocation of thioredoxin into the nucleus
where it directly associates with Ref-1 [47•]. Such a
pathway may provide an explanation for how activity is
restored for transcription factors which are activated by
oxidative stress but require a reduced state to bind DNA.
ROS as mediators of disease
A variety of human diseases have been linked to an
overproduction of ROS. The growing realization that
oxidants may function in signaling pathways may, in
turn, cause a re-evaluation of the pathways and sources
of oxidant production linked to human disease. As
an example, as shown in Figure 3, both human and
animal data suggests that prior to the development of
atherosclerotic plaque, the vessel wall appears to produce
an increase in superoxide anions [48]. The increase in
O2− has been postulated to contribute to atherogenesis
by a variety of mechanisms including the inactivation
of NO, the oxidation of low-density lipoprotein and the
stimulation of NF-κB. In addition, a variety of clinical
and epidemiological evidence supports a protective role for
antioxidants in cardiovascular disease [49].
Could the pathways described above be activated in
the pre-atherosclerotic vessel? Does hypertension or
hypercholesterolemia activate Ras or Rac1 proteins, and
is the observed increase in ROS simply a marker of a
continual activation of these small GTPases? Although
clearly speculative, it is interesting to note that one
characteristic of atherosclerotic vessels is an increase in
turbulent flow. Recent evidence using an in vitro model
of this condition, namely cells exposed to an increase in
251
Figure 3
LDL
VCAM-1
O2– + NO
Endothelial dysfunction
NF-κB
Current Opinion in Cell Biology
Putative role of superoxide anions in the development of
atherosclerosis. Increased O2− can interact with, and thereby
inactivate, locally produced NO, a clinical syndrome known as
endothelial dysfunction. Similarly, it can lead to the oxidation of
low-density lipoprotein (LDL) cholesterol. Finally, the activation of
NF-κB which is regulated by ROS and which in turn regulates the
expression of various adhesion molecules (e.g. VCAM-1), has also
been linked to atherogenesis.
sheer stress, demonstrate that endothelial cells appear to
respond to an increase in flow through the activation of
small GTP-binding proteins [50,51] and the subsequent
induction of a host of gene products which appear to
regulate the redox state of the cell [52,53]. In addition,
in vivo infusion of agents linked to hypertension, such
as angiotensin II, result in the increased expression of
p22phox and augmented NADPH oxidase activity in the
vessel wall, as well as a stimulation of O2- production
[54,55•]. As such, the further delineation of these pathways
may allow for the development of specific oxidase
inhibitors that would presumably be significantly more
specific and effective than the current class of scavenging
antioxidants. Such inhibitors may have applications not
only in atherogenesis, but in a host of human diseases that
have been linked to ROS and which include reperfusion
injury, Alzheimer’s and aging.
Conclusions
This review has dealt with the hypothesis that reactive
oxygen species such as O2− and H2O2 act as intracellular
signaling molecules. The recent discovery of a role for key
cellular proteins such as Ras and p53 in controlling oxidant
levels suggest that the redox state is actively regulated.
Indeed, the alteration of protein function by oxidants may
be in many ways analogous to phosphorylation except that
protein modification no longer occurs on specific serine
or tyrosine residues but instead on redox-sensitive amino
acids such as cysteine and histidine. Delineation of the
pathways regulated by oxidants may have fundamental
implication in the control of diverse cellular events
including transcription, growth and death. Significant work
needs to be done in identifying direct targets of ROS and
the relevant sources of oxidant production. Such work will
hopefully provide some understanding of how specificity
can be obtained using ROS as signal transducers. In
252
Cell regulation
addition to providing fresh insight into a host of biological
questions, these studies might provide a new direction
and therapeutic approach to a wide assortment of human
diseases, in which ROS are implicated.
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Acknowledgements
I wish to thank N Epstein and S Gutkind for helpful comments on
this manuscript and D Bar-Sagi for sharing results before publication. In
addition, I thank my collaborators K Irani and PJ Goldschmidt-Clermont as
well as members of my laboratory for stimulating discussions.
17.
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